1. Field of the Invention
The present invention relates to heptahelix receptors and methods of using them to identify compounds that have biological and pharmaceutical activity. More specifically, this invention relates to the discovery of ligands for a heptahelix receptor that was previously an unknown (“orphan”) receptor, and to the development of assays to screen for other compounds that have inhibitory or activating effects on the receptor.
2. Description of Related Art
Some extracellular molecules, such as dietary substances, small molecule metabolites, hormones, vasodilators, and neurotransmitters affect the cellular activity of certain, select cells within an organism to bring about localized or systemic changes in the physiology of the organism. The effects brought about by these molecules often require interaction between the extracellular molecules and cell surface proteins present on target cells. Receptors are among the cell surface proteins that respond to these extracellular molecules, and initiate the events that lead to changes in cellular activity and, thus, physiological changes in the organism.
Cell surface receptors are membrane-spanning proteins that bind extracellular signaling molecules or otherwise sense changes in the extracellular environment, then initiate one or more signal transduction pathways to effect a cellular response. Cell surface receptors are classified based on the particular type of pathway that is induced. One of the most intensely studied classes of cell surface receptor is the class known as heptahelix receptors. These receptors bind extracellular molecules and couple that binding to binding, at the intracellular portions of the receptor, to intracellular effector proteins, such as guanine nucleotide binding regulatory proteins, which are typically referred to as G-proteins.
In general, G-protein transmembrane signaling pathways consist of three proteins: receptors, G-proteins, and effectors. As discussed above, the receptors are membrane-bound proteins that couple an external stimulus to an internal stimulus. G-proteins, which, most often, are the intermediaries in transmembrane signaling pathways of heptahelix receptors, are heterodimers that consist of alpha, beta, and gamma subunits. G-proteins transfer the signal or stimulus present on the intracellular portion of the receptor to the effectors. The effectors bring about the physiological change intended, typically through an alteration in the transcriptional activity of certain genes. Thus, in summary, signal transduction through G-protein coupled, heptahelix receptor systems proceeds as follows: 1) signal transduction is initiated by ligand (i.e., extracellular molecule) binding to a specific portion of the receptor; 2) binding of ligand causes an intracellular signal to be generated by the receptor, likely through a conformational change in the receptor; 3) the intracellular portion of the receptor binds GDP-bound G-protein, resulting in dissociation of the GDP from the G-protein; 4) the G-protein then binds GTP at the site where GDP was bound, thus activating the G-protein; 5) activated G-protein dissociates from the receptor and activates an effector protein, which regulates the intracellular levels of one or more specific second messengers; 6) hydrolysis of GTP to GDP, catalyzed by the G-protein itself, returns the G-protein to its basal, inactive form, which can bind to the receptor.
Heptahelix receptors are known to share certain structural similarities and regions of homology. For example, G-protein coupled receptors have seven hydrophobic stretches of about 20-30 amino acids each, bracketed outside the first and last and interrupted between each by at least eight hydrophilic regions of variable length. It is generally accepted that each of the seven hydrophobic regions forms an alpha helix that spans the cell membrane, and that the intervening and surrounding hydrophilic regions form alternating intracellular and extracellular loops. The seven transmembrane regions are designated TM1, TM2, TM3, TM4, TM5, TM6, and TM7. The third intracellular loop between TM5 and TM6 is believed to be the intracellular domain responsible for interaction with G-proteins. Furthermore, most receptors have single conserved cysteine residues in each of the first two extracellular loops, which form disulfide bonds that are believed to stabilize the functional protein structure. It is thought that phosphorylation and/or lipidation (e.g., palmitylation or farnesylation) of cysteine residues on some heptahelix receptors can influence signal transduction. Most heptahelix receptors contain potential phosphorylation sites within the third cytoplasmic loop and/or the carboxy terminus. The ligand binding sites of heptahelix receptors are believed to comprise a hydrophilic socket formed by several transmembrane domains surrounded by hydrophobic residues.
It is well established that many medically significant biological processes are mediated by heptahelix receptor signaling pathways. For example, the heptahelix receptor family includes dopamine receptors, which bind to neuroleptic drugs used for treating psychotic and neurological disorders, as well as receptors for calcitonin, noradrenaline, endothelin, cAMP, adenosine, acetylcholine, serotonin, histamine, thrombin, kinin, follicle stimulating hormone, opsins and rhodopsins, and odorants.
Heptahelix receptors thus play important physiological roles. Accordingly, there are many potential pharmacological uses for compounds that interact with and modulate the activity of heptahelix receptors. Indeed, a number of compounds that are known to be useful in treating various diseases in animals, including humans, are thought to exert their beneficial effects through interactions with heptahelix receptors. Examples include the blockade of β-adrenergic receptors in cardiac disease, the blockade of serotonin receptors in migraine, the blockade of leukotriene receptors in inflammatory diseases, and the blockade of purine receptors to exert thrombolytic effects. In fact, most of the drugs now in clinical use exert their effects by interfering, in some fashion, with heptahelix receptors.
Unfortunately, an understanding of the pharmacology of compounds that interact with heptahelix receptors, and the ability to rapidly identify compounds that specifically interact with them to provide desired therapeutic effects, have been hampered by the lack of rapid and sensitive methods or assays to identify those compounds. In vitro methods are commonly used now to screen and identify candidate compounds. For example, a rapid, sensitive method for identifying ligands for heptahelix receptors was recently developed (Kotarsky et al., 2001). The availability of this method has enabled researchers to screen a large number of compounds to identify those that show promise as pharmaceuticals, or at least to identify core compounds that can be modified to yield pharmaceuticals.
Lipids are small molecules that can be found both intracellularly and extracellularly in animals, including humans. Lipids provide energy to the cell and contribute to cellular components including organelles and the plasma membrane. Thus, they serve a role in both the metabolism of cells and in the physical structure of cells.
The simplest lipids are the fatty acids. Fatty acids are long chain hydrocarbons attached to carboxyl groups. They are classified generally in two categories—saturated and unsaturated. Saturated fatty acids derive their name from the fact that every carbon atom in their chains is fully saturated (i.g., each internal carbon is bound to two hydrogen atoms and the terminal non-carboxyl carbon is part of a methyl group). Accordingly, all the bonds between the carbons in the chain are single bonds. In contrast, unsaturated fatty acids contain carbons that are not saturated with hydrogens—they contain carbon-carbon double bonds. Naturally occurring unsaturated fatty acids contain double bonds that are in the cis configuration, whereas artificially produced unsaturated fatty acids contain double bonds that are in the trans configuration. Trans fatty acids are found in margarine and other foods, and have been linked with heart disease. The most common saturated fatty acids in humans are palmitic acid (C16) and stearic acid (C18). The most common unsaturated fatty acid in humans is oleic acid (C18).
Fatty acids serve as the building blocks for various higher lipids, such as triglycerides (triacyl glycerols), phospholipids, steroids (such as cholesterol), and lipoproteins (such as chylomicrons, the very low density lipoproteins (VLDLs), the low density lipoproteins (LDLs), and the high density lipoproteins (HDLs)). Diabetics typically have elevated chylomicron and VLDL levels and depressed levels of LDLs, whereas people with high cholesterol diets typically have elevated LDL and VLDL levels. Persons with gout typically have elevated LDL and VLDL levels. Alcoholics typically have elevated chylomicron, LDL, and VLDL levels. Furthermore, elevated trans fatty acids in a diet have been shown to be linked to hypercholesterolemia, atherosclerosis, coronary artery disease, and coronary heart disease. Thus, although essential nutritional components, fatty acids are known risk factors in cardiovascular and metabolic diseases. (See, for example, Unger, 2002.)
During the last decade, it has become evident that different classes of lipids serve as chemical messengers in the body. (Chawla et al., 2001.) For example, the eicosanoids prostaglandin and leukotriene, which are derived from the fatty acid arachidonic acid (C20), are now known to act as extracellular signaling molecules in vasodilation, muscle contraction, and chemotaxis, and are involved in allergy and anaphylaxis. Likewise, oleic acid has recently been shown to be involved in triggering neutrophil aggregation and neutrophil adherence to epithelial cells, and has thus been implicated in fat embolisms that cause acute respiratory distress. (Mastrangelo et al., 1998.) Interestingly, oleic acid has also been linked to the low incidence of atherosclerotic disease in Mediterranean countries, where intake of oleic acid is relatively high. The effect of oleic acid appears to take place through modulation of expression of endothelial leukocyte adhesion molecules. (See, for example, Massaro et al., 1999.) The nutritional and metabolic effects of short-chain fatty acids have been well described. (Linder, 1991).
It is now clear that lipids play an essential and important role as both dietary components and disease factors. Accordingly, there is a need in the art to identify mechanisms by which lipids and more complex molecules derived from them are metabolized or exert their specific effects on diseases or disorders. By identifying and understanding the mechanisms of lipid metabolism and lipid interaction with various diseases and disorders, new drugs and treatment regimens for the diseases and disorders can be developed and delivered to those suffering from, or at risk of developing, the diseases or disorders.